Characterization of type A and type B CCK receptor binding sites in rat vagus nerve

Abstract We employed quantitative receptor autoradiography to analyze pharmacological properties of 125 I-Bolton Hunter cholecystokinin (CCK-8)-labeled binding sites in sections of rat cervical vagus nerve that had been ligated 24 h prior to extraction. Binding densities were detected in segments of nerve proximal and distal to the ligature. Analysis was confined to proximal segments. Saturation and competitive binding studies were carried out using sulphated CCK-8 and two selective CCK receptor antagonists: MK-329, to define type-A (CCK A ) binding sites; and, L-365,260, to define type-B (CCK B ) binding sites. Sulphated CCK-8 was the most potent inhibitor of vagal 125 I-CCK binding (IC 50 = 2nM). Nonlinear curve fitting analysis of the CCK binding data favored the presence of a single class of vagal CCK receptorsKD i = 1nM). However, both MK-329 (IC 50 = 18nM) and L-365,260 (IC 50 = 45nM) completed for vagal 125 I-CCK binding indicating the presence of CCK A and CCK B binding sites. Co-analysis of the antagonist binding data suggested that CCK A and CCK B receptors were transported in equal concentration within the vagus. MK-329 bound with high affinity to CCK A sites (K i = 3nM) and low-affinity to CCK B sites (K i = 462nM) while L-365,260 bound with high affinity to CCK B sites (K i = 10nM) and low-affinity to CCK A sites (K i = 775nM). These same ligands were used to characterize the specificity of 125 I-CCK binding in the medial and lateral divisions of the nucleus of the solitary tract (NTS), two regions innervated by primary vagal afferents carrying CCK receptors. Sulphated CCK-8 was the most potent inhibitor of 125 I-CCK binding in both regions of the NTS (IC 50 = 0.6nM, medial;IC 50 = 0.4nM, lateral). In the medial NTS, MK-329 was a potent inhibitor (IC 50 = 21.8nM) while MK-329 was a weak inhibitor (IC 50 = 341nM) of 125 I-CCK binding. In contrast, in the lateral NTS, L-365,260 was a potent inhibitor (IC 50 = 21.8nM) while MK-329 was a weak inhibitor (IC> 1,0000 nM) of 125 I-CCK binding. These results are consistent with the existence of two populations of vagal afferent fibers projecting to different regions of the NTS. One, containing CCK A receptors, projects principally to the medial NTS, while the other containing CCK B receptors, projects principally to the lateral NTS.